Studies were conducted to express and purify the extracellular domain and subdomains of human IFN alpha receptor (HuIFN-R) and to generate antibodies for the investigation of signal transduction mechanisms. Expression and Purification of HuIFN-R: The gene encoding the extracellular domain of HuIFN-R was expressed as fusion with glutathione-S-transferase (GST). The GST-receptor was isolated from inclusion bodies in the absence (soluble form) and presence (insoluble form) of 8 M urea. Overall recovery was approximately 1 mg/liter of cell culture. Both insoluble and soluble forms inhibited the antiviral and antiproliferative activities of 5 -10 U of IFN alpha and showed a Kd value of approximately 10-9 in receptor-ligand binding assays. Soluble Form of GST-Receptor: Induction of protein expression with 0.1 mM IPTG at 30 degrees C, sequential disruption with french press in Tris pH 9.0 buffer containing DTT, EDTA, PMSF and CHAPS (or Triton) released HuIFN- R soluble form from inclusion bodies. Expression and Isolation of HuIFN-R Subdomains: Three subdomains (residues 1-103, 93-260 and 224-401) were expressed in PRSET vector and served to identify several hybridoma colonies which reacted with specific regions found in each fragment. Cloning and monoclonal antibody production from those colonies are near completion. Antibody Production: Polyclonal antibodies against GST-receptor produced 25 - 30% inhibition of both IFN alpha binding to receptor and IFN biological activity. Testing of inhibition of IFN biological properties by monoclonal antibodies is in progress. Findings from these investigations will significantly enrich our understanding of the mechanisms by which interferons and other cytokines modulate immune responses and control viral and neoplastic growth through their receptors.